Fourteen out of the 59 genera were represented with less than 10 isolates. The phylogenetic composition of the cultivable community isolated in our study in the presence of antibiotics did not differ considerably from the common profile of

any aquatic environment [33–35]. The selection towards Gammaproteobacteria is a well known Selleckchem PF 2341066 plating bias of aquatic bacterial communities [36]. When the isolates from antibiotic-containing plates were compared with isolates growing on drug free ZoBell medium no striking differences between major genera were observed (Peeter Laas, unpublished data). Figure 1 Unrooted Bayesian Etomoxir nmr phylogenetic tree of the 760 isolates using the 16S rRNA gene sequences. The scale bar represents 1.0 expected changes per nucleotide position. The nodes are color-coded according to the antibiotics used to isolate the strains, but the area is not proportional to the number of isolates from that antibiotic. The width of the node is in proportion to the number of isolates in each node. The antibiotics are designated as follows: Amp – ampicillin, Cam – chlorapmhenicol, Kan – kanamycin, Nor – norfloxacine, Tet – tetracycline. The numbers indicate genera as follows: 1 – Flexibacteriaceae, 2 – Sphingobacterium, 3 – Pedobacter, 4 – Flavobacterium, 5 – Elizabethkingia, 6 – Chryseobacterium, 7 – Deinococcus, 8 – Brachybacterium, 9 – Microbacteriaceae, 10 – Cellulomonadaceae, 11 – Micrococcaceae, 12 – Nocardiaceae,

13 – Nocardioidaceae, 14 – Sanguibacter, 15 – Bacillales, 16 – Sphingomonadaceae, Sirtuin inhibitor 17 – Hyphomicrobiaceae, 18 – Caulobacteraceae, 19 – Ensifer, 20 – Alcaligenaceae, 21 – Oxalobacteriaceae, 22 – Incertia cedis, 23 – Comamonadaceae, 24 – Aeromonas, 25 – Enterobacteriaceae, 26 – Acinetobacter, 27 – Pseudomonas,

28 – Xanthomonadaceae. We had two sampling stations, one upstream of a town with 100,000 inhabitants (Tartu, Estonia) and the other downstream. No statistically significant differences in the phylogenetic affiliation and AR patterns were observed when Tau-protein kinase bacteria isolated from upstream or downstream were compared (data not shown). Characterization of antibiotic resistance As our isolates showed a wide variety of growth rates and growth curve shapes, the standard MIC test could not be applied. Instead we grew the isolates in 96-well plates in the presence and absence of antibiotic. The cultures were grown at 20°C without shaking and the OD was measured at 16, 20, 24, 40 and 64 h. All five antibiotics used for the isolation of the strains were used to test the level of resistance of all of the isolates in the collection. As the collection contained a large number of Pseudomonas strains, and increased carbapenem resistance is a problem in Estonian medical settings [37], we included a member of this group of antibiotics, meropenem, in the resistance testing. The growth of an antibiotic-sensitive strain is inhibited by the drug, thus leading to a lower optical density.